Metabolic perturbations and key pathways associated with the bacteriostatic activity of Clitoria ternatea flower anthocyanin fraction against Escherichia coli

Clitoria ternatea flowers are rich in anthocyanins and possess various biological activities. Specifically, the antibacterial mechanism of action of C. ternatea anthocyanins remains unknown and was investigated in Escherichia coli . A time–kill assay was used to assess the antibacterial activity and the metabolic perturbations in E. coli were investigated utilizing liquid chromatography–mass spectrometry (LC-MS)-based metabolomics. Pathway analyses were carried out for metabolites showing ≥2-fold changes. The anthocyanin fraction remarkably reduced the growth of E. coli at 4 h by 95.8 and 99.9 % at minimum inhibitory concentration (MIC) and 2× MIC, respectively. The anthocyanin fraction (MIC) had a bacteriostatic effect and was shown to have perturbed glycerophospholipids (1-acyl-sn-glycero-3-phosphoethanolamine, phosphatidylglycerol, diacylglycerol and cardiolipin), amino acids (valine, tyrosine and isoleucine) and energy (ubiquinone and NAD) metabolites at 1 and 4 h. This study demonstrated significant metabolic perturbations of the glycerophospholipid, amino acid and energy metabolism, with these being the key pathways involved in the bacteriostatic activity of anthocyanins from C. ternatea, which may have promise as bacteriostatic agents for E. coli -related infections.


INTRODUCTION
Although Escherichia coli, Gram-negative bacteria of the family Enterobacteriaceae, comprise a common, normal part of the flora in the human gut, they have become a concern because of their pathogenic strains, which are known to cause severe infections, along with the increase in the emergence of multidrug resistance globally [2]. E. coli have been identified as significant causative pathogens in bloodstream and urinary tract infections worldwide in community and healthcare settings [3]. They have displayed high rates of resistance to antibiotics such as aminoglycosides, aminopenicillins, fluoroquinolones and third-generation cephalosporins, further complicating and limiting treatment options [3].
The rise in antimicrobial resistance means there is a need for new antimicrobial agents or alternative therapeutic methods/ strategies to target infectious diseases. Plants are a rich source of compounds with diverse functional groups in which many are yet to be explored for their therapeutic potential [4]. The Clitoria ternatea ('blue pea' or 'butterfly pea') plant belongs to the family Fabaceae and is known to grow in various countries in Asia and South and Central America [5,6]. The leaves, flowers and roots of this plant have been used since ancient times to treat a variety of ailments and health conditions besides reports on their various biological activities (e.g. antioxidant, antidiabetic, anti-inflammatory, antifungal and antiproliferative/anticancer) [5,6].

OPEN ACCESS
C. ternatea flowers have ornamental value and are also used as a food colouring agent due to the presence of the compounds known as anthocyanins [7]. The crude extract of the flower has been reported to show antibacterial activity against various bacterial strains (e.g. E. coli, Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus), which has mainly been attributed to its major compounds, flavonols and anthocyanins [8,9].
Metabolomics is a comprehensive analysis investigating large-scale metabolite levels in biological systems. It can be further categorized as targeted (identifies and quantifies selected metabolites of interest, e.g. enzymes, substrates or compounds of a particular pathway) or untargeted (involves measuring all metabolites in a desired biological experimental system, which usually generates a new hypothesis based on the outcome) approach, depending on the nature of the study [10,11]. Metabolomics is increasingly being used to elucidate the mechanism of action of drugs in a biological system for drug discovery and development [12]. Our previous study [13] obtained an anthocyanin fraction from this flower to determine the antibacterial activity against various pathogenic bacterial strains. Antibacterial activity was observed against B. subtilis, E. coli and Bacillus cereus. As E. coli is a pathogen of importance, the present study aimed to determine the antibacterial activity and the mechanism of action of the anthocyanin fraction against E. coli through a metabolomics study.

METHODS Preparation and extraction of samples
Clitoria ternatea cv. Double Blue flowers (freshly harvested) were procured from a horticultural nursery in Subang Jaya, Malaysia. C. ternatea flower petals were plucked carefully for use in the study, while the remaining parts were discarded. Before extraction, the petals were cut into smaller pieces. Ethanol extracts of the fresh flower material were prepared by immersing the flowers in 50 % ethanol with a solid to liquid ratio of 1 : 20 (g ml −1 ) for 3 h at room temperature (25 °C) with constant shaking. Extracts were then vacuum-filtered, and a rotary evaporator was used to concentrate the solution under vacuum at 45 °C. The concentrated solution was freeze-dried and stored at −80 °C until further analysis.

Semi-purification of crude extract by Amberlite XAD-16 column chromatography
The anthocyanin fraction was obtained according to the method in a previous study [14]. Distilled water (100 ml) was used to dissolve the freeze-dried extract of C. ternatea flowers (5 g), adjusted to pH 2 using 1M HCl followed by partitioning with ethyl acetate (100 ml) to facilitate the removal of flavonols. The aqueous fraction (containing anthocyanins) was collected and again partitioned two more times with ethyl acetate. The aqueous fraction (containing anthocyanins) was concentrated to a volume of 10 ml under vacuum at 37 °C in a rotary evaporator. The aqueous fraction (containing anthocyanins) was subjected to further purification using Amberlite XAD-16 column chromatography. A chromatography column [S24/29, 25(D)×300 mm (l)] with a sintered glass disc (porosity 0) and a stopcock with a PTFE key were used for the open column chromatography. The Amberlite XAD-16 adsorbent was immersed in methanol overnight prior to loading to ¾ of the column height. Briefly, 1 l of purified water was used to rinse the column followed by activation with 0.5 l of aqueous sodium hydroxide (2%) solution. Acidified water (1 l) was used to wash the column to condition it to pH 3. The concentrated aqueous fraction (10 ml) of the anthocyanin fraction was loaded onto the column, which was then rinsed with acidified water (0.3 l at pH 3) to remove phenolic acids at a flow rate of 10 ml min −1 . Anthocyanins were then eluted with acidified methanol [95 : 5, methanol : acidified water (pH 2), v/v]. The methanol (contains anthocyanins) fraction was freeze-dried and stored at −80 °C until further analysis. The extraction yield of the anthocyanin fraction using Amberlite XAD-16 column chromatography was 18.2 %, as reported in our previous study [13].

Time-kill assay of E. coli ATCC 25922
The culture of E. coli ATCC 25922 was prepared from frozen stock (−80 °C) on tryptic soy agar plates and incubated at 37 °C for 18 h. A single colony was obtained and inoculated into brain heart infusion (BHI) broth (10 ml), incubated at 37 °C for 18 h in a shaking water bath (180 r.p.m.). The overnight culture diluted with BHI broth (1 : 100) was grown to obtain an OD 600nm of 0.50 to achieve an early logarithmic growth phase (i.e. ~10 8 c.f.u. ml −1 ), followed by dilution (1 : 100) in 20 ml of BHI broth alone (control group) and BHI broth containing C. ternatea anthocyanin fraction at ½ MIC (5 mg ml −1 ), MIC (10 mg ml −1 ) and 2× MIC (20 mg ml −1 ) for 1, 4 and 24 h. One millilitre of sample was taken at 0, 1, 4 and 24 h and serial dilutions were performed according to the pour plate method as described below to determine bacterial growth (c.f.u. ml −1 ). The formula below was used to count the percentage reduction in the total viable count of c.f.u.:

Statistical analysis
The experiments above were performed in independent triplicates and the results were expressed as the mean value±standard deviation. The data were analysed using one-way analysis of variance (ANOVA) followed by post-hoc Tukey's test to analyse the statistical difference exhibited by E. coli ATCC 25922 in terms of growth in untreated (control) and treated groups [½ MIC (5 mg ml −1 ), MIC (10 mg ml −1 ) and 2× MIC (20 mg ml −1 )]. The significance was set at P<0.05 using SPSS 23 software (IBM, New York, USA).

Bacterial culture preparation for metabolome analysis
Bacterial isolates of E. coli ATCC 25922 were sub-cultured from −80 °C stocks onto tryptic soy agar. A single colony was obtained and inoculated in BHI broth (10 ml). It was incubated overnight at 37 °C with constant shaking (180 r.p.m.). The overnight culture was diluted by 1 : 100 (2.5 ml was inoculated into 500 ml) of BHI broth and grown to obtain an OD 600 of 0.5 (mid-exponential growth phase) at 37 °C with constant shaking (180 r.p.m.). Subsequently, 20 ml of mid-exponential culture (OD 600 = 0.5) was collected into a 50 ml Falcon tube (this was used as 0 h). For each sample, 50 ml of the mid-exponential culture was treated with the anthocyanin fraction of C. ternatea (AF) resuspended in BHI broth at MIC (10 mg ml −1 ), while the untreated group served as a control sample. Three biological replicates of each group were prepared. The bacterial cultures (20 ml) of treated and untreated groups were collected at 1 and 4 h.

Extraction and liquid chromatography-mass spectrometry (LC-MS) analysis of cellular metabolites
Cellular metabolites were extracted using a previously reported method [17,18]. The bacterial cultures (20 ml) of treated and untreated groups at 0, 1 and 4 h were immediately transferred into ice-cold 50 ml Falcon tubes. To stop the metabolic processes, the samples were quenched rapidly in a dry ice/ethanol bath for 30 s. The culture was then normalized to 0.5 at OD 600 (cell counts were at 10 8 c.f.u. ml −1 ) using fresh BHI broth. The normalized culture (10 ml) was centrifuged (3220 g at 4 °C for 10 min) and the cell pellet was obtained. Ice-cold 0.9 % sodium chloride (2 ml) was used to wash the cell pellet twice followed by centrifugation (3220 g at 4 °C for 5 min). The residue after centrifugation was removed. After discarding the supernatant, 0.5 ml of dry-ice cold chloroform : methanol : water (1 : 2 : 0.8, v/v) extraction solvent containing internal standard at 1 µM of 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS) was added to resuspend the cells and facilitate the extraction of the intracellular metabolites. Liquid nitrogen was used to freeze the samples and allowed to thaw on ice to promote the release of intracellular metabolites. The freeze-thaw process was performed thrice and followed by centrifugation (3220 g at 4 °C for 10 min). The supernatant was obtained and transferred to 1.5 ml centrifuge tubes and centrifuged (14400 g at 4 °C for 10 min). The samples were stored at −80 °C prior to LC-MS analysis. The samples (3 µl) were analysed on a 1290 Infinity LC system coupled to a 6520 Accurate-Mass Q-TOF mass spectrometer with a dual ESI source (Agilent, Santa Clara, CA, USA) coupled to a HILIC-Z column (2.7 µm, polymeric, 100×2.1 mm; Infinitylab Poroshell, Agilent). The run was performed in positive and negative ion modes at a resolution of 35 000. The detection range was 85 to 1275 m/z. The mobile phases were 20 mM ammonium acetate (A) and acetonitrile (B) at a flow rate of 0.3 ml min −1 . The applied condition was as follows: 20 % of solvent A and 80 % B to 50 % B over 15 min, and to 5 % B at 18 min. The system was washed with 95 % A and 5 % B for 3 min followed by 20 % A and 80 % B for 8 min to re-equilibrate the column. Nitrogen was used as the desolvation gas, at 300 °C and a flow rate of 60 l h −1 , and He gas was used as the damping gas, with a declustering potential of 40 eV, a collision energy of 5 eV and a collision cell entrance potential of 10 eV. The samples were run in a single batch of LC-MS to avoid batch-to-batch variability [19].

Antibacterial activity of the C. ternatea anthocyanin fraction against E. coli ATCC 25922
The antimicrobial activity of C. ternatea anthocyanin fraction against a laboratory strain E. coli ATCC 25922 utilizing the agar dilution method (ADM) showed the MIC value to be 10 mg ml −1 at 24 h in our previous study. The MIC is defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a micro-organism after overnight incubation [13]. The concentration of the fraction was found not to affect the viability of E. coli at ≤5 mg ml −1 (viability >95 %). A time-kill assay was performed with E. coli treated at ½ MIC (5 mg ml −1 ), MIC (10 mg ml −1 ) and 2× MIC values (20 mg ml −1 ) of C. ternatea anthocyanin fraction (Fig. 1). The time-kill assay showed that the fraction at ½ MIC value did not affect the bacterial growth over a period of 24 h. The reduction of bacterial growth (c.f.u. ml −1 ) by treatment groups was determined at respective time points compared to the untreated group. At 1 h, the bacterial growth (c.f.u. ml −1 ) was 11.2×10 5 , 6.0×10 5  The impact of regional differences of the compound composition of the C. ternatea crude flower extract is unknown, as the composition was not reported in these studies. The crude flower extract is known to have a much lower content of anthocyanins, suggesting that the high content of anthocyanins (composed mainly of ternatin B2/B3, ternatin D1 and ternatin D2) in the fraction used in this study is responsible for the antibacterial activity. This can also be supported by the findings observed in  The MIC (10 mg ml −1 ) of the C. ternatea anthocyanin fraction was determined in our previous study using the agar dilution method [13]. However, in this current study, the time-kill study was performed using the suspension method (in a flask with large volume as the metabolomics study requires a larger amount of culture to proceed with study analysis) with constant shaking, which may further accelerate the growth of bacterial culture, contributing to the effect observed at 24 h in the current study.
In this current study, the C. ternatea anthocyanin fraction at MIC and 2×MIC value at 4 h displayed a bacteriostatic effect, as the reduction of log c.f.u. ml −1 of E. coli ATCC 25922 ( Fig. 1) at 4 h for MIC or 2× MIC was not greater than a 3 log 10 -fold decrease in c.f.u.
[25], which shows that not all bacterial cells are killed at 4 h and the bacteriostatic effect is not maintained at 24 h. A similar pattern has also been observed in another study with other natural compounds from garlic and guava [26]. It is also suggested that the antibacterial activity exhibited by C. ternatea anthocyanin fraction may be contributed by the synergistic activity of the anthocyanins in the fraction.
Bacteriostatic agents can be defined as agents that are able to prevent the growth of bacteria without causing bacterial cell death directly [27,28]. Drugs with a bacteriostatic effect are more advantageous than those that are bactericidal as they may help to prevent drug resistance [27][28][29]. Observations have shown bacteriostatic agents to cause a lower incidence of toxic shock and more tolerable side effects clinically [27]. Apart from that, from our previous study [13], although the crude extracts of C. ternatea flower displayed toxicity at doses above 156.3 µg ml −1 against HEK-293 cells (human embryonic kidney cells), the findings from the acute toxicity study in rats show the extract to be rather safe for consumption.
An acute toxicity study was performed using albino Wistar rats, which were treated orally with 50 % ethanol extract of C. ternatea flowers at 2000 mg kg −1 body weight. The treatment group showed no signs of mortality or abnormality and there was no significant difference in the haematological values compared to the control untreated group, which indicates no acute toxicity of C. ternatea However, further studies investigating the toxicity in preclinical models would be essential.

Metabolic profile of E. coli ATCC 25922 treated with C. ternatea anthocyanin fraction
The mechanism of action of the anthocyanin fraction of C. ternatea for its bacteriostatic activity against E. coli is not known and was further investigated utilizing untargeted metabolomics analysis to determine key signalling pathways that may be responsible for the antibacterial effect observed. Untargeted metabolomics was used, as it is not known which metabolites are or would be affected upon treatment with C. ternatea anthocyanin fraction. LC-MS-based metabolomics was used for analysing polar and nonpolar small molecules [31]. The metabolic perturbations of the C. ternatea anthocyanin fraction against E. coli were determined at its MIC (10 mg ml −1 ) at 1 and 4 h treatment (Fig. 2). Approximately 800 metabolites were detected in the control (untreated) and treated groups at 1 and 4 h and were subjected to PCA followed by statistical analysis utilizing the t-test. A clear clustering between treatment and control groups was observed for the metabolite profile at 1 (PC1=60.7 %) and 4 h (PC1=48.6 %) using PCA (Fig. 3a). The log 2 fold change (≤−1 or ≥1) was determined for the metabolites that were significantly perturbed in the treatment group compared to the control. In total, the number of significantly affected metabolites at 1 h was 81/126 (up/down) and 53/116 (up/down) at 4 h. (Fig. 2b). These metabolites were then subjected to identification utilizing the online databases ECMDB, KEGG and LIPID MAPS followed by pathway analysis. The significantly perturbed metabolites were from the metabolism of lipids, amino acids and energy, which are further discussed below.

Perturbation of phospholipid and glycerophospholipid metabolism by C. ternatea anthocyanin fraction
Glycerophospholipids are important components of the outer membrane (a dual membrane composed of lipopolysaccharide in the outer leaflet and glycerophospholipids in the inner leaflet) of Gram-negative bacteria, which is essential to protect the bacteria from toxic compounds and harsh environmental conditions. The inner membrane (symmetrical bilayer) of the bacteria, by contrast, is known to be composed of phospholipids, which are essential for the maintenance of barrier permeability and in support of its membrane proteins [32].
The treatment of C. ternatea anthocyanin fraction at 10 mg ml −1 (MIC) was found to have exerted growth inhibition and significantly perturbed the glycerophospholipid metabolites of E. coli at 1 and 4 h ( A larger number of glycerophospholipid metabolites were perturbed at 1 h compared to 4 h (Fig. 3). The perturbations observed in the glycerophospholipid metabolites indicate membrane damage induced by C. ternatea anthocyanin fraction treatment. This may trigger enhanced synthesis and transport of glycerophospholipids to re-establish the outer membrane barrier of a bacterium [34]. This is also supported by the significant increase in the level of CL metabolites. CL metabolites play an essential role in remodelling of the bacterial outer membrane, as CL is also important for bacterial structural integrity and cell function [35].
E. coli membranes are mainly composed of PE (75%) followed by PG (20%) and CL (5%) [36,37]. Changes in the environmental conditions or exposure to antibacterial agents can impose stress on bacteria, triggering certain responses to enable their survival and requiring major metabolic reprogramming [36]. The results obtained on the phosphoglycerolipid metabolite levels upon treatment with the anthocyanin fraction showed disruption of the optimum conditions required for membrane integrity, which may have caused alterations in cell physiology, which also compromises the integrity of the cell.
Compromised integrity of the cell membrane upon treatment could have led to a series of different cellular effects, such as cellular envelope structure modification, disruption of energy transduction metabolic pathways and impairment of membrane and macromolecule synthesis [37]. This can be supported by the findings reported in another study for the antibacterial effect of the anthocyanins of blueberries. The anthocyanins elicited bacterial cell membrane damage, resulting in leakage of intracellular materials as well as facilitating entry into the cell. It also further disrupted the tricarboxcylic acid (TCA) cycle and energy metabolism of the bacteria [38]. The results obtained in our study showed a remarkable effect of C. ternatea anthocyanin fraction, which triggered the perturbation of various phospholipids to cause disruption of membrane integrity to inhibit the growth of E. coli.

Perturbation and inhibition of amino acid and energy metabolism of C. ternatea anthocyanin fraction
The disruption of bacterial membrane integrity by C. ternatea anthocyanin fraction could have facilitated its entry into the cell, affecting the stability of the intracellular environment and leading to the perturbations of the level of amino acid metabolites. The amino acids leucine (log 2 FC=−1.04 and −1.74), isoleucine (log 2 FC=−1.26 and −2.03), valine (log 2 FC=−1.86 and −2.27), tyrosine (log 2 FC=−1.64 and −1.96) and phenylalanine (log 2 FC=−1.24 and −1.91) were reduced at both 1 and 4 h of treatment, respectively. There was a higher reduction of the metabolite levels of amino acids at 4 h compared to 1 h (Fig. 4a). The effect on the level of amino acid metabolites was higher later at 4 h as compared to the effect on glycerophospholipid metabolites, which was higher at 1 h (Fig. 3). This may be a subsequent effect that occurs after the perturbation of glycerophospholipids upon entry of anthocyanins into the cells.
Amino acids are the basic building blocks of proteins, and are also biomolecules that are essential in the biological functions of micro-organisms, such as metabolism, growth and survival of cells, formation of cell walls, division of cells and bacterial cell communication (quorum sensing). Enzymes, being composed of amino acids and classified as proteins, play a crucial role in facilitating the resistance of microbes to antimicrobial drugs [39]. The glycolytic pathway, the TCA cycle and the pentose phosphate pathway are oxidative pathways to obtain energy. The reduction of amino acid metabolite levels may reflect it being used as a substitute for carbon source due to the bacteriostatic effect of the anthocyanin fraction. This may be an adaptation mechanism of bacteria to thrive in environmental conditions to maximize their chance of survival [40, 41].
The TCA cycle is involved in the oxidation of different metabolic intermediates derived from the catabolism of lipids, carbohydrates and certain amino acids. For each turn of the TCA cycle, three molecules of NAD + are reduced to NADH and one molecule of FAD is reduced to FADH 2 . These molecules then transfer their energy to the electron transport chain, which in turn releases energy so that it can be converted to adenosine triphosphate (ATP) as an energy source for bacteria [42]. Disruption of the metabolic activity of the bacterial TCA cycle causes the weakening of cellular respiration, making the energy supply inadequate, which can lead to cell death [43]. At the examined treatment concentration (MIC=10 mg ml −1 ) (Fig. 4a), C. ternatea anthocyanin fraction affected the TCA cycle, where it was found to have reduced the metabolite level of succinate (log 2 FC=−1.28 and −3.8) at both 1 and 4 h, respectively, with a higher reduction at 4 h. The reduction of succinate by the anthocyanin fraction of C. ternatea may have disrupted the energy metabolism of the electron transfer chain by reducing the electron transfer to ubiquinone as the rate of the electron transfer from ubiquinone to NAD is limited by the low content of succinate, which serves as the main reductant of ubiquinone [41,44].
In E. coli, ubiquinone is a lipid that plays an important role in the electron transfer chain to generate energy [45]. Upon treatment with the anthocyanin extract, the levels of NAD (log 2 FC=−1.33 and −2.96) and ubiquinone-4 (log 2 FC=−1.58 and −1.94) declined significantly at 1 and 4 h, respectively, with a higher reduction at 4 h (Fig. 4b). In conclusion, the anthocyanin fraction of C. ternatea has a potential bacteriostatic effect against E. coli. To the best of our knowledge, this is the first study to reveal significant metabolic perturbations of C. ternatea anthocyanin fraction against E. coli, revealing its mechanism of action. The anthocyanins in the fraction could have acted synergistically in exerting the bacteriostatic effect by perturbing glycerophospholipid metabolism, amino acid and energy metabolism, with these being the major pathways involved in the effect of C. ternatea anthocyanin fraction against E. coli.
Structure-activity relationship studies would be beneficial to understand the effect of the structural groups responsible for or contributing to the bioactivity. Future studies looking into combination treatment of C. ternatea anthocyanin fraction with clinical agents may be beneficial; its potential action as a bacteriostatic agent might enhance the antibacterial effect of clinical agents. Assessment of the impact on bacterial membrane integrity using imaging studies would be highly beneficial. It is also essential to carry out further studies to determine its effect on clinical and multidrug-resistant E. coli strains. The anthocyanin fraction could potentially be used to reduce E. coli-related infections. However, further studies investigating toxicity in pre-clinical models are also essential. This study provides novel metabolomic information on the mechanism of action of C. ternatea anthocyanin fraction to reduce E. coli-related infections.

Funding information
This work was funded and supported by the School of Science, Monash University Malaysia.  Comments: I would like to thank the authors for their efforts in applying the comments and suggestions in their manuscript. However, there are still some minor points to amend. Please find them below: ·L393-394: "Amino acids in the form of enzymes" reads as if amino acids had catalytic activity themselves. Please rephrase to properly convey that enzymes are proteins, and thus are formed by amino acids ·L394-396: The glycolytic pathway, the TCA cycle and the pentose phosphate pathway are oxidative pathways to obtain energy, reduced coenzymes and metabolic precursors that are streamed to other metabolic branches, such as amino acid anabolic reactions (as illustrated in Figure 4A). However, this reads as if those oxidative pathways were used for making amino acids. Please rephrase this. ·L467: I would really like to commend the authors for availing their data to the public. As an Open Research Platform, we really value these efforts for transparency. However, some more explanation on the content that is shared would be needed in this "Data summary" section. Additionally, it would be ideal if the content that is shared had any sort of legend that allowed the reader to precisely know what they will find in the different spreadsheets. If this modification cannot be made directly on the shared document, please add the explanation related to the different data sheets in this section as well.

RESPONSE TO EDITOR'S COMMENTS:
1)Although it was suggested by Reviewer 1 and fixed to some extent, a clean-cut definition of the difference between bactericidal and bacteriostatic activity, which happens to be important for the interpretation of the results, is not entirely achieved. Please provide it supported with the appropriate references.

Response:
The reduction of log CFU/mL of E. coliATCC 25922 (Figure 1) at 4 h for MIC or 2 × MIC is not greater than 3 log 10 -fold decrease in colony forming units (Khader et al., 2020) and therefore the anthocyanin fraction of C. ternateaflower is bacteriostatic. A correction has been made and supporting references have now been included (lines 258 -263). 2) In line with the previous comment, as the conclusion is that the flower fraction of study is specifically bactericidal, I strongly recommend to consider a change of the title including this, rather than the generic "antibacterial activity"

Response:
A correction has been made and confirmed the anthocyanin fraction to be bacteriostatic. The title has now been changed and specific to "bacteriostatic":

Metabolic perturbations and key pathways associated with the bacteriostatic activity of Clitoria ternatea flower anthocyanin fraction against Escherichia coli
3) In comment 19 from Reviewer 1, there is a discussion on the toxicity of the anthocyanin fraction on eukaryotic cells and rats. However, this is not included in the manuscript. I strongly suggest introducing this in the manuscript where appropriate, as it will contribute to enrich the discussion.

Response:
This discussion has now been included in the manuscript (lines 270 -280).

Response:
The brackets have now been removed 5) L230: use "respectively" to match each CFU count to each MIC-based concentration

Response:
"respectively" has now been added to match each CFU count to each MIC-based concentration (lines 231 and 233).

Response:
Citations for the different databases have now been included (lines 209 and 216)

7)
Statistical analysis (L154-158): state which statistical test is applied to which dataset, as well as the significance threshold (p-value), for example in the respective figure legends

Response:
This information has now been included (lines 156-160). Statistical analysis tests for metabolomics section are on lines 212-215.

8) L381
: "Amino acids being the basic building blocks of proteins, are also biomolecules that are essential in the biological functions of microorganisms…" Please correct the grammar in this sentence

Response:
The grammar in the sentence has now been changed (lines 390-394).

9)
In the interest of transparency and reproducibility, consider making the whole metabolomics dataset publicly available, either as supplementary material or uploading to the appropriate database and citing it in the manuscript. Please refer to the Microbiology Society Open Data Policy for more information (https://www.microbiologyresearch.org/open-data)

10)
An exhaustive revision of the references is needed, especially with respect to appropriateness and format. Examples of this are: o L309-314: reference 25 is used for explaining the differences between OM and IM. However, this is explained in the introduction of reference 25, it is not a result of that work. Please use the original refences when possible and appropriate throughout the manuscript.

Response:
The reference has now been changed (line 323).
o L314-317: reference 26 has to do with the trafficking of phospholipids and CL in mitochondria, not in bacteria. Please refer to a more content-appropriate article to support this information.

Response:
The reference has now been changed (line 326).
o L390-394: The TCA cycle is not directly involved in the oxidation of lipids, carbohydrates and amino acids. It is involved in the oxidations of different metabolic intermediates that derive from the catabolism ofthose via multiple branches of the metabolism (e.g. pyruvate, alpha-ketoglutarate). Please correct and be as precise as possible in general statements throughout the manuscript. Furthermore, reference 34, used for supporting this, refers to the TCA cycle in skeletal muscle, whilst there is plenty of information directly related to the TCA cycle in bacteria.

Response:
The sentence has been corrected and the reference has now been changed (lines 400-401 and line 405).
o L394-395: reference 35 is unrelated to the TCA and/or bacterial respiration, and thus to this statement

Response:
The reference has now been changed (lines 406 and 421).

Response:
The reference has now been included (line 425).

VERSION 2
is concise and well written, and congratulations to the authors for these exciting and interesting findings. While it is clear that exposure to the anthocyanin fractions has an impact of E. coli, the precise nature of this impact needs some additional clarity and the focus of the findings of the metabolomic data needs to be sharpened.

1)-
The terms bacteriostatic and antibacterial are both used to describe the effects of C. ternateathroughout the paper. The authors should clarify the distinction between the two terms and select.

Response:
The antibacterial activity of C. ternateaanthocyanin fraction was investigated, the observed effect was found to be bacteriostatic as growth was observed at 24 h treatment time and the metabolomics study was done at the MIC only. The term antibacterial has been now changed to bacteriostatic throughout as determined from the time-kill assay.
2)-It needs to be made clear in the main text to the reader if the strain of E. coli is being tested is of clinical origin or is multidrug resistant. If the strain is not of clinical origin or MDR, do the authors think the extract will be effective against these more recalcitrant strains.

Response:
The E. colistrain used in this study was a laboratory strain. This has now been mentioned in the beginning of the "Discussion" section (lines 219-220). An additional statement has now been included in the "Conclusion" section suggesting further studies to be conducted on clinical and MDR strains (lines 431-432).

3)-
The authors need to explore the true nature of the effect of this fraction in more detail. The MIC was previously determined as 10mg/ml, however from Figure 1 it is quite clear that at 24 hours, the E. coli have overcome the inhibitory effect of the fraction and have recovered to the same CFU as the negative control. The other issue with this figure is that at 2xMIC there is a clear killing effect as stated in the text the CFU drops from 7.4 x 10^5 to 2 x10^2. This suggests that the fraction may be bactericidal. The other issue with this figure is that in all conditions it appears that there is an increase in bacterial growth from 0-1 hour, how can this be explained given the inhibitory nature of the fraction, you would expect the effect to be immediate. Given this is a fraction however, rather than a pure compound there could be synergistic and antagonistic interactions taking place at once. This section would benefit from a growth curve analysis with readings every 15 minutes.

Response:
The MIC (10 mg/mL) of C. ternateaanthocyanin fraction was determined in our previous study (Jeyaraj et al., 2022) using the agar dilution method. However, in this current study, the time-kill study was done using the suspension method (in a flask with large volume as the metabolomics study requires a larger amount of culture to proceed with study analysis) with constant shaking which may further accelerate the growth of bacterial culture thus contributing to the effect seen at 24 h in the current study. In this current study, C. ternateaanthocyanin fraction at MIC value at 4 h displays a bacteriostatic effect while at 2 × MIC potentially displays bactericidal effect with a high possibility that not all bacterial cells are killed at 4 h as a bacteriostatic or bactericidal effect is not maintained at 24 h. It is also suggested that the antibacterial activity exhibited by C. ternateaanthocyanin fraction may be contributed by the synergistic activity of the anthocyanins in the fraction. It would be beneficial for further studies to be conducted on the effect of the C. ternateaanthocyanin fractionon the growth curve analysisat shorter time intervals to further understand its bacteriostatic/bactericidal effect at MIC and 2 × MIC. This paragraph has now been included in the manuscript (lines 254-267).

4)-
The authors base a significant component on the MIC that was previously determined in Jeyaraj et al.,2022. However, this MIC was determined in an agar dilution method, and as much of this study is based in broth cultures, it would be very beneficial to conduct an MIC assay in liquid broth. The benefit of this also is that there may be regional or temporal impacts on anthocyanin concentration that would lead to differences in the MIC from batch to batch. Findings should also be put in context with Uma et al., 2009 where the crude flower extract had an MIC of between .25-10 mg/mL.

Response:
The use of broth cultures in this current study as compared to the previous study findings on MIC value using agar dilution method, and the essential need for further studies on growth curve analysis has now been mentioned in main text (lines 254-267). We were not able to determine the MIC values using the broth dilution method due to the strong extract colour which masks the detection of the absorbance value at the higher concentration range tested. Based on our findings from the agar dilution method (Jeyaraj et al., 2022), the growth inhibition effect was not seen for the crude extracts on E. coliup to 40 mg/mL which correlates to findings from other studies but was different to that reported by Uma et al., 2009. It is unknown on the impact of regional differences of the compound composition of the C. ternateacrude flower extract as it was not reported in their study. This has now been included in the manuscript (lines 235-241).

5)-
For the time kill assays -what was the anthocyanin fraction resuspended in? Is a vehicle control needed for the assay? Response:

13)-
The study would be greatly strengthened by some validation of the impact on membrane integrity using perhaps live cell imaging or a kit such as LIVE/DEAD BacLight Bacterial viability kit.

Response:
We highly agree it would be beneficial to validate on the impact on membrane integrity using imaging studies. However, we do not have the resources to carry this out. A suggestion for further studies looking at this aspect has now been included in lines 430-431.
14)-Line 354 how does this higher reduction at 4 hours as compared to 1 hour compared with the previous section on the impact on glycerophospholipids?

Response:
This could be possibly due to the subsequent effects that may occur after the perturbation of glycerophospholipids upon entry of anthocyanins into the cells. This has now been included in lines 377-381. The disruption of the bacteria membrane integrity (glycerophospholipids) by C. ternateaanthocyanin fraction could have facilitated its entry into the cell which affected the stability of the intracellular environment leading to the perturbations of the level of amino acid metabolites. Thus, the effect on the level of amino acid metabolites was higher later at 4 h as compared to glycerophospholipids (lines 371-373).
15)-Authors should show the ubiquinol-6 and -8 at 1 hr to give a full picture of the results.

Response:
The metabolite levels of ubiquinone-6 and ubiquinol-8 were decreased at 4 h but not at 1 h thus it was not shown in Figure 4. A statement for the effect not seen at 1 h for ubiquinone-6 and ubiquinol-8 has now been included in line 407-408.
16)-Greater weight should be added to the effect on the TCA cycle as this is strengthened by the previous work which the authors have cited by Sun et al., 2018.

Response:
More information has now been included in lines 412-417.
17)-If standards of Ternatins are available to purchase, authors should consider this as a control for their assays.

Response:
Currently standards for ternatins are not available for purchase.

18)-
For the bacterial culture preparation for metabolome analysis -why was a different agar (nutrient agar) used to grow the colonies than in the time kill assay?

Response:
This has now been corrected and changed to Tryptic Soy agar in lines 161-162.

19)-
The authors state in line 402-404 that the anthocyanin fraction could be used to reduce E. colirelated infections however this does not align with previous work published by the authors which states that the anthocyanin rich extract displays toxicity at doses above 156.3 µg/mL against HEK-293 cells (Jeyaraj et al.,2022, Figure 3), which is much lower the MIC described in this work. Greater context needs to be added here.

Response:
From the previous study (Jeyaraj et al., 2022), although the crude extracts of C. ternateaflower displayed toxicity at doses above 156.3 µg/mL against HEK-293 cells, the findings from the acute toxicity study in rats show the extract to be rather safe for consumption. An acute toxicity study was done using albino Wistar rats which were treated orally with 50% ethanol extract of C. ternateaflowers at 2000 mg/kg body weight. The treatment group showed no signs of mortality or abnormality and there was no significant difference in the hematological values compared to the control untreated group which indicates no acute toxicity of C. ternateaflower extracts up to 2000 mg/kg (Srichaikul, 2018). "The anthocyanin fraction could be potentially used to reduce E. coli-related infections. However further studies investigating the toxicity in pre-clinical models would be essential. " This statement has now been included in lines 432-434.

Response:
This statement and reference have been removed. "The anthocyanins in the fraction could have acted in synergism for exerting the bacteriostatic effect by perturbing the glycerophospholipid metabolism, amino acid and energy metabolism being the major pathways involved for the effect of C. ternateaanthocyanin fraction in E. coli. Structure-activity relationship studies would be beneficial to understand the effect of the structural groups responsible or contributing to the bioactivity". This has now been included in lines 423-427.

Response:
This was referring to findings from our previous study which determined the antibacterial activity of the crude extract and anthocyanin-rich fraction of C. ternateaagainst various Gram-positive and Gram-negative bacteria. This statement intended to show the higher antibacterial activity (based on MIC value) of the anthocyanin-rich fraction when compared to the crude extract. Figure 1, Line 237 -Are the replicates technical or biological?

Response:
The replicates are biological and has now been included in line 276.

REVIEWER 2:
The work has some value and significance, although the advantages and disadvantages should be considered: Advantages: the work investigates mechanism of action of anthocyanins fraction against E. coli -it is new Disadvantages: The phytochemical analysis of the obtained fraction was not presented, hence indeed we don't know what were the active compounds used in the experiment.
The methodology lacks many details, and in general is not precise, strongly needs clarification, also in the section related to bacterial metabolites identification.
In the discussion: it is suggested to rethink the presented observations in regard to the study "Cell Metab. 2019 Aug 6; 30 (2) The authors of the manuscript under evaluation suggest that decreased energy production results in bacterial cell death, however in the earlier paragraphs they stated that observed effect of anthocynanins was bacteriostatic (lines 225-226). It is contradictory. Also, if you analyse the suggested work, you will find the information that decreased metabolic activity in bacteria is strictly related to bacteriostatic effect of antibiotics. Hence it is mandatory to re-evaluate observations to make correct conclusions.

Response:
The amendments have been made based on the remarks below.
Other remarks:
8)It is not stated anywhere what solvent was used to dissolve the frozen anthocyanins for the antimicrobial experiments.

Response:
BHI broth was used to dissolve the anthocyanin fraction. This information has now been included in lines 132 and 168. 9)Could you please clarify the volume of the experimental culture: line 159 states 50 ml, while line 167 states 20ml.

Response:
For each sample, 50 mL was prepared for treated and untreated groups with 3 biological replicates. From the 50 mL, 20 mL was collected at 1 h and 20 mL was collected from the remaining source at 4h. This has now been further explained in lines 163-171.

10)
Line 170: what was used to normalize cultures?

Response:
Fresh BHI broth was used to normalize the cultures. This has now been included in line 178.

11)
Line 179: was the residue after centrifugation removed?

Response:
The residue after centrifugation was removed. This has now been included in line 181.

12)
Lines 181-185: the parameters of ion source and detector should be given.

Response:
This has now been included in lines 197-199.

13)
Line 193: authors reported that retention time was used for identification, so were authentic standards used? because in databases retention time is rather not reported. It also would be good to specify the allowed mass difference which was applied during identification (delta ppm). Can you please specify how the internal standard was used in the calculations?

Response:
Authentic standards were not used. We used m/z values to assist or identify a particular metabolite which appears across all samples (control and treated groups at 1 and 4 h) and compared to the available online databases (E. coliMetabolome Database (ECMDB), Kyoto Encyclopedia of Genes and Genomes (KEGG) and LIPID MAPS) to determine the putative metabolites. The statement on using retention time to identify metabolites in database has now been removed.
14)Line 219: it is hard to believe that you can dissolve 100mg of plant extract in 1 ml! Rather suspension can be obtained.

Response:
Further clarification and explanation have now been included to compare results obtained by other studies in lines 235-239.

Response:
The statement "high content of anthocyanins (composed mainly of Ternatin B2, Ternatin D1 and Ternatin D2) in the fraction used in this study responsible for the antibacterial activity" is based on our previous study (Jeyaraj et al., 2022)  16)Lines 241-251: it is not clear why the discussion about the structure of compounds present in used fraction is placed in the if you analyse the suggested work, you will find the information that decreased metabolic activity in bacteria is strictly related to bacteriostatic effect of antibiotics. Hence it is mandatory to re-evaluate observations to make correct conclusions. Other remarks: Please make it clear: was the extraction yield 50%? The 10 g of plant material was used to prepare extract , than 5g of the extract was used for semi-purification. Was it possible to obtain 5g of the extract from 10g of plant material? Line 107: what was used for pH adjustment? Line 112: what was the size (weight) of the amberlite resin? Line 115: was water used to dissolve sample? Line 117: how the flow rate was controlled? What was the final yield of the purified anthocyanins fraction? Line 128: What was the volume of cultures treated with anthocyanins? It is not sated anywhere what solvent was used to dissolve the frozen anthocyanins for the antimicrobial experiments. Could you please clarify the volume of the experimental culture: line 159 states 50 ml, while line 167 states 20ml. Line 170: what was used to normalize cultures? Line 179: was the residue after centrifugation removed? Lines 181-185: the parameters of ion source and detector should be given Line 193: authors reported that retention time was used for identification, so were authentic standards used?, because in databases retention time is rather not reported. It also would be good to specify the allowed mass difference which was applied during identification (delta ppm). Can you please specify how the infernal standard was used in the calculations? Line 219: it is hard to believe that you can dissolve 100mg of plant extract in 1 ml! Rather suspension can be obtained. Line 220-223: Can you support this statement with reference? The other authors report also ternatin A1, A2, A3, B1, B2, B3, B4, preternatin A3 (https:// www3. e-kenkyu. com/ bpb-reports-online-journal/ uploads/ manuscript/ file/ 137/ 4_ 136. pdf). Lines 241-251: it is not clear why the discussion about the structure of compounds present in used fraction is placed in the section "Metabolic profile of E. coli (ATCC 25922) treated with C. ternatea anthocyanin fraction" Lines: 394-395: this statement about glycoside residues is not supported by any proof in the evaluated work, firstly, the composition of the used fraction was not determined, secondly, the glucose moiety can be substituted by p-coumaroyl or malonyl residues.

Please rate the manuscript for methodological rigour Poor
Please rate the quality of the presentation and structure of the manuscript Satisfactory To what extent are the conclusions supported by the data? Partially support

Anonymous.
Date report received: 22 December 2022 Recommendation: Major Revision Comments: 1. Methodological rigour, reproducibility and availability of underlying data Good but the MIC section and the effect on growth need to explored in greater detail. 2. Presentation of results Good, particularly for the metabolomics although the data does not appear to have been uploaded to a metabolomics database which would support transparency. 3. How the style and organization of the paper communicates and represents key findings Good 4. Literature analysis or discussion Quite a few sections need to be more appropriately referenced as highlighted above. 5. Any other relevant comments In this manuscript, the authors present findings of the anthocyanin fractions from Clitoria ternatea flowers as having a bacteriostatic effect on E. coli. Through metabolome analysis they show the involvement of disrupted membrane integrity, perturbed glycerophospholipid and energy metabolism as the key pathways involved in bacteriostatic effects of the plant. The manuscript is concise and well written, and congratulations to the authors for these exciting and interesting findings. While it is clear that exposure to the anthocyanin fractions has an impact of E. coli, the precise nature of this impact needs some additional clarity and the focus of the findings of